Insulin is the peptide hormone that regulates glucose uptake and metabolism. The two types of diabetes mellitus are associated either with an inability to produce insulin because of destruction of the pancreatic islet cells (Homo-Delarche, F. & Boitard, C., 1996, Immunol. Today 10: 456–460) or with poor glucose metabolism resulting from either insulin resistance at the target tissues, or from inadequate insulin secretion by the islets or faulty liver function (Taylor, S. I., et al., 1994, Diabetes, 43: 735–740).
Insulin-like growth factors-1 and 2 (IGF-1 and 2) are structurally related to insulin, but are more important in tissue growth and development than in metabolism. They are primarily produced in the liver in response to growth hormone, but are also produced in most other tissues, where they function as paracrine/autocrine regulators. The IGFs are strong mitogens, and are involved in numerous physiological states and certain cancers (Baserga, R., 1996, TibTech 14: 150–152).
Epidermal growth factor (EGF) is a small polypeptide cytokine that is unrelated to the insulin/IGF family. It stimulates marked proliferation of epithelial tissues, and is a member of a larger family of structurally-related cytokines, such as transforming growth factor α, amphiregulin, betacellulin, heparin-binding EGF and some viral gene products. Abnormal EGF family signalling is a characteristic of certain cancers (Soler, C. & Carpenter, G., 1994 In Nicola, N. (ed)“Guidebook to Cytokines and Their receptors”, Oxford Univ. Press, Oxford, pp 194–197; Walker, F. & Burgess, A. W., 1994, In Nicola, N. (ed) “Guidebook to Cytokines and Their receptors”, Oxford Univ. Press, Oxford, pp 198–201).
Each of these growth factors mediates its biological actions through binding to the corresponding receptor. The IR, IGF-1R and the insulin receptor-related receptor (IRR), for which the ligand is not known, are closely related to each other, and are referred to as the insulin receptor subfamily. A large body of information is now available concerning the primary structure of these insulin receptor subfamily members (Ebina, Y., et al., 1985 Cell 40: 747–758. Ullrich, A., et al., 1985, Nature 313: 756–761; Ullrich, A. et al., 1986, EMBO J 5: 2503–2512; Shier, P. & Watt, V. M., 1989, J. Biol. Chem. 264: 14605–14608) and the identification of some of their functional domains (for reviews see De Meyts, P. 1994, Diabetologia 37: 135–148; Lee, J. & Pilch, P. F. 1994 Amer. J. Physiol. 266: C319–C334; Schaffer, L. 1994, Eur. J. Biochem. 221: 1127–1132). IGF-1R, IR and IRR are members of the tyrosine kinase receptor superfamily and are closely related to the epidermal growth factor receptor (EGFR) subfamily, with which they share significant sequence identity in the extracellular region as well as in the cytoplasmic kinase domains (Ullrich, A. et al., 1984 Nature 309: 418–425; Ward, C. W. et al., 1995 Proteins: Structure Function & Genetics 22: 141–153). Both the insulin and EGF receptor subfamilies have a similar arrangement of two homologous domains (L1 and L2) separated by a cys-rich region of approximately 160 amino acids containing 22–24 cys residues (Bajaj, M., et al., 1987 Biochim. Biophys. Acta 916: 220–226; Ward, C. W. et al., 1995 Proteins: Structure Function & Genetics 22: 141–153). The C-terminal portion of the IGF-1R ectodomain (residues 463 to 906) is comprised of four domains: a connecting domain, two fibronectin type 3 (Fn3) repeats, and an insert domain (O'Bryan, J. P., et al., 1991 Mol Cell Biol 11: 5016–5031). The C-terminal portion of the EGFR ectodomain (residues 477–621) consists solely of a second cys-rich region containing 20 cys residues (Ullrich, A. et al., 1984, Nature 309: 418–425).
Little is known about the secondary, tertiary and quaternary structure of the ectodomains of these receptor subfamilies. Unlike the members of the EGFR subfamily which are transmembrane monomers which dimerise on binding ligand, the IR subfamily members are homodimers, held together by disulphide bonds. The extracellular region of the IR/IGF-1R/IRR monomers contains an α-chain (˜703 to 735 amino acid residues) and 192–196 residues of the β-chain. There is a ˜23 residue transmembrane segment, followed by the cytoplasmic portion (354 to 408 amino acids), which contains the catalytic tyrosine kinase domain flanked by juxtamembrane and C-tail regulatory regions and is responsible for mediating all receptor-specific functions (White, M. F. & Kahn, C. R. 1994 J. Biol. Chem. 269: 1–4). Chemical analyses of the receptor suggest that the α-chains are linked to the β-chains via a single disulphide bond, with the IR dimer being formed by at least two α—α disulphide linkages (Finn, F. M., et al., 1990, Proc. Natl. Acad. Sci. 87: 419–423; Chiacchia, K. B., 1991, Biochem. Biophys. Res. Commun. 176, 1178–1182: Schaffer, L. & Ljungqvist, L., 1992, Biochem. Biophys. Res. Comm. 189: 650–653; Sparrow, L. G., et al., 1997, J. Biol. Chem. 47: 29460–29467).
Although the three-dimensional (3D) structures of the ligands EGF, TGF-alpha (Hommel, U., et al., 1992, J. Mol. Biol. 227:271–282), insulin (Dodson, E. J., et al., 1983, Biopolymers 22:281–291), IGF-1 (Sato, A., et al., 1993, Int J Peptide Protein Res 41:433–440) and IGF-2 (Torres, A. M., et al., 1995, J. Mol. Biol. 248:385–401) are known, and numerous analytical and functional studies of ligand binding to EGFR (Soler, C. & Carpenter, G., 1994 In Nicola (ed) “Guidebook to Cytokines and Their receptors”, Oxford Univ. Press, Oxford, pp 194–197), IGF-1R and IR (see De Meyts, P., 1994 Diabetologia, 37:135–148) have been carried out, the mechanisms of ligand binding and subsequent transmembrane signalling have not been resolved.
Ligand-induced, receptor-mediated phosphorylation is the signalling mechanism by which most cytokines, polypeptide hormones and membrane-anchored ligands exert their biological effects. The primary kinase may be part of the intracellular portion of the transmembrane receptor protein, as in the tyrosine kinase receptors (for review see Yarden, Y., et al., 1988, Ann. Rev. Biochem. 57:443–478) or the Ser/Thr kinase receptors (Alevizopoulos, A. & Mermod, N., 1997, BioEssays, 19:581–591) or may be non-covalently associated with the cytoplasmic tail of the transmembrane protein(s) making up the receptor complex, as in the case of the haemopoietic growth factor receptors (Stahl, N., et al., 1995, Science 267:1349–1353). The end result is the same, ligand binding leads to receptor dimerization or oligomerization or a conformational change in pre-existing receptor dimers or oligomers, resulting in activation by transphosphorylation, of the covalently attached or non-covalently associated protein kinase domains (Hunter, T., 1995, Cell, 80:225–236).
Many oncogenes have been shown to be homologous to growth factors, growth factor receptors or molecules in the signal transduction pathways (Baserga, R., 1994 Cell, 79:927–930; Hunter, T., 1997 Cell, 88:333–346). One of the best examples is v-Erb (related to the EGFR). Since overexpression of a number of growth factor receptors results in ligand-dependent transformation, an alternate strategy for oncogenes is to regulate the expression of growth factor receptors or their ligands or to directly bind to the receptors to stimulate the same effect (Baserga, R., 1994 Cell, 79:927–930). Examples are v-Src, which activates IGF-1 R intracellularly; c-Myb, which transforms cells by enhancing the expression of IGF1R; and SV40 T antigen which interacts with the IGF-1R and enhances the secretion of IGF-1 (see Baserga, R., 1994 Cell, 79:927–930 for review). Cells in which the IGF-R has been disrupted or deleted cannot be transformed by SV40 T antigen. If oncogenes activate growth factors and their receptors, then tumour suppressor genes should have the opposite effect. One good example of this is the Wilm's tumour suppressor gene, WT1, which suppresses the expression of IGF-1R (Drummond, J. A., et al., 1992, Science, 257:275–277). Cells that are driven to proliferate by oncogenes undergo massive apoptosis when growth factor receptors are ablated, since, unlike normal cells, they appear unable to withdraw from the cell-cycle and enter into the G0, phase (Baserga, R., 1994 Cell, 79:927–930).
The insulin-like growth factor-1 receptor (IGF-1R) is one of several growth-factor receptors that regulate the proliferation of mammalian cells. However, its ubiquitousness and certain unique aspects of its function make IGF-1R an ideal target for specific therapeutic interventions against abnormal growth, with very little effect on normal cells (see Baserga, R., 1996 TIBTECH, 14:150–152). The receptor is activated by IGF1, IGF2 and insulin, and plays a major role in cellular proliferation in at least three ways: it is essential for optimal growth of cells in vitro and in vivo; several cell types require IGF-1R to maintain the transformed state; and activated IGF-1R has a protective effect against apoptotic cell death (Baserga, R., 1996 TIBTECH, 14:150–152). These properties alone make it an ideal target for therapeutic interventions. Transgenic experiments have shown that IGF-1R is not an absolute requirement for cell growth, but is essential for the establishment of the transformed state (Baserga, R., 1994 Cell, 79: 927–930). In several cases (human glioblastoma, human melanoma; human breast carcinoma; human lung carcinoma; human ovarian carcinoma; human rhabdomyosarcoma: mouse melanoma, mouse leukaemia; rat glioblastoma; rat rhabdomyosarcoma; hamster mesothelioma) the transformed phenotype can be reversed by decreasing the expression of IGF-1R using antisense to IGF-1R (Baserga, R., 1996 TIBTECH 14:150–152); or by interfering with its function by antibodies to IGF-1R (human breast carcinoma; human rhabdomyosarcoma) or by dominant negatives of IGF-1R (rat glioblastoma; Baserga, R., 1996 TIBTECH 14:150–152).
Three effects are observed when the function of IGF-1R is impaired: tumour cells undergo massive apoptosis which results in inhibition of tumourogenesis; surviving tumour cells are eliminated by a specific immune response; and such a host response can cause a regression of an established wild-type tumour (Resnicoff, M., et al., 1995, Cancer Res. 54:2218–2222). These effects, plus the fact that interference with IGF-R function has a limited effect on normal cells (partial inhibition of growth without apoptosis) makes IGF-1R a unique target for therapeutic interventions (Baserga, R., 1996 TIBTECH 14:150–152). In addition IGF-1R is downstream of many other growth factor receptors, which makes it an even more generalised target. The implication of these findings is that if the number of IGF-1Rs on cells can be decreased or their function antagonised, then tumours cease to grow and can be removed immunologically. These studies establish that IGF-1R antagonists will be extremely important therapeutically.
Many cancer cells have constitutively active EGFR (Sandgreen, E. P., et al., 1990, Cell, 61:1121–135; Karnes, W. E. J., et al., 1992, Gastroenterology, 102:474–485) or other EGFR family members (Hines, N. E., 1993, Semin. Cancer Biol. 4:19–26). Elevated levels of activated EGFR occur in bladder, breast, lung and brain tumours (Harris, A. L., et al., 1989, In Furth & Greaves (eds) The Molecular Diagnostics of human cancer. Cold Spring Harbor Lab. Press, CSH, NY, pp 353–357). Antibodies to EGFR can inhibit ligand activation of EGFR (Sato, J. D., et al., 1983 Mol. Biol. Med. 1:511–529) and the growth of many epithelial cell lines (Aboud-Pirak E., et al., 1988, J. Natl Cancer Inst. 85:1327–1331). Patients receiving repeated doses of a humanised chimeric anti-EGFR monoclonal antibody showed signs of disease stabilization. The large doses required and the cost of production of humanised monoclonal antibody is likely to limit the application of this type of therapy. These findings indicate that the development of EGF antagonists will be attractive anticancer agents.